Pharmacologic therapies for uterine leiomyomas are hampered by our limited knowledge regarding the origin and evolution of these tumors, as well as the degree of molecular heterogeneity. Leiomyomas, also known as fibroids, are the most common benign tumors of uterine smooth muscle cells and are a major cause of morbidity among American women. Previous studies have suggested that fibroids are monoclonal in origin. However, histological and cell sorting analyses of leiomyomas have shown cellular heterogeneity, with presence of fibroblasts in addition to the smooth muscle cells in leiomyoma tumours. Whole exome approaches from our group and others have identified mutations in the mediator complex subunit 12 (MED12) in approximately 70% of LM patients, indicating that MED12 mutant cells might give rise to leiomyomas. We generated a mouse model that showed leiomyoma tumor formation in uteri that express Med12 mutation. We will utilize our mouse models to begin the study of leiomyoma early origins and interact with other Projects of this P50 application, to define molecular heterogeneity of leiomyomas and their relation to the tumor genotype. Our studies will focus to: 1) understand the onset and progression of Med12 mutation positive leiomyomas, 2) the evolution of genomic instability in uterine leiomyomas, and 3) understand the relationship between leiomyoma genotype and molecular heterogeneity that may impact leiomyoma recurrence rates and non-responsiveness to therapy. Our mouse model and preliminary findings will complement studies of other investigators in this P50 application. We will provide Dr. Bulun's Project 1 with our mouse model and in vivo preliminary data that Med12 interacts with the progesterone pathway, as well as complement his studies on different human leiomyoma cell types with our own studies in mice and humans. Dr. Chakravarti's Project 3 will benefit from our single cell sequencing on MED12 positive, HMGA2 positive, and MED12/HMGA2 negative leiomyomas and will complement his epigenetic studies on HMGA2 positive leiomyomas. Together, the studies proposed by us and our collaborators will provide great insights into the pathophysiology of uterine LM. Our studies will identify origin of Med12 positive leiomyomas, determine if genotype drives molecular phenotype and cellular heterogeneity, with a goal of driving targeted therapy for leiomyomas.